Library Construction and RNA Sequencing
Total RNAs was extracted from the root samples of four soybean genotypes
under control and stress conditions using TRIzol reagent by following
the manufacturer’s protocols (Invitrogen, Carlsbad, CA, USA). The RNA
quantity and integrity was checked using Nano-Drop Spectrometer (ND-1000
Spectrophotometer, Thermo Scientific) and Agilent Bioanalyzer 2100
(Agilent Technologies, Santa Clara, CA, US), respectively. To select the
best quality RNA an integrity value ≥7 was used for library
construction. Total RNA of 2 μg from each sample was used for the
construction of paired-end libraries using Illumina TruSeq RNA Sample
Preparation Kit (Illumina, San Diego, CA, USA), in accordance with the
manufacturer’s instructions. Qubit® 2.0Fluorometer (Life Technologies,
USA) was used to quantify purified libraries, and are authenticated by
Agilent 2100-bioanalyzer (Agilent Technologies, USA) to validate the
fragment size and concentration. Furthermore, libraries were diluted up
to 10 Pm for cBot clustering and RNA-Seq was performed using Illumina
HiSeq 2500 platform by Shanghai Biotechnology Corporation.